DMPA associated with vaginal bacterial diversity in Kenyan sex workers. (A-D) Three α-diversity metrics were used to compare bacterial richness and evenness within the vaginal microbiota of Kenyan sex workers not on hormonal contraceptives (proliferative phase of menstrual cycle, N=22), on oral contraceptives (N=14) and on DMPA (N=22), from the same geographical region. Significant differences in observed species were initially observed during Kruskal–Wallis tests, however no significant differences were subsequently identified during post-hoc testing (A). Chao1 richness was significantly higher in the VMB of sex workers on DMPA versus those on OCPs (P≤0.05; Kruskal–Wallis tests), yet no significant differences were observed between NH sex workers and those on DMPA (B). Sex workers on DMPA had the greatest bacterial diversity at all levels of rarefaction (P≤0.05; Kruskal–Wallis tests), followed by women not on hormonal contraceptives, and those on oral contraceptives (C). Sex workers on DMPA had significantly greater bacterial diversity in their vaginal microbiota than those not on hormonal contraceptives, at all depths of rarefaction (P≤0.05; Mann–Whitney U-test) (D). ns, not significant. *P≤0.05, **P≤0.01. Data are mean±s.e.m.

Contraceptives and Lactobacillus dominance and clustering. (A) The top 20 bacterial genera in the vaginal microbiota were plotted by relative abundance as taxa bar charts and compared between sex workers who were not on hormonal contraceptives (proliferative phase of the menstrual cycle, N=22), on oral contraceptives (N=14) or on DMPA (N=22). Each bar represents the vaginal microbiota of one woman. Each color represents a different genus of bacteria, as indicated. Species of Lactobacillus are indicated in gray/patterns. Vaginal microbiota are ordered left to right in descending order of the relative abundance of Lactobacillus. The proportion of women with Lactobacillus-dominant VMB was assessed between groups using several different cut-points of relative abundance (Table 1). At the highest cut-off chosen (≥98% lactobacilli) there were significantly more women with Lactobacillus-dominant VMB in the NH (proliferative phase) and OCP groups than in the DMPA group, suggesting that a high estrogen state is associated with a greater abundance of lactobacilli, and supporting our results demonstrating enhanced Shannon diversity in women on DMPA (Fig. 1C,D). Asterisk indicates resolved to family level. (B) The PCoA demonstrated the β-diversity of the vaginal microbiota at the operational taxonomic unit (OTU) level based on the Bray–Curtis dissimilarity matrix. The vaginal microbiota did not cluster by method of contraception, but clustered based on the CSTs previously described (Ravel et al., 2011), by the dominance of Lactobacillus in the vaginal microbiota. Sex workers dominated by L. crispatus (CST I) are circled in blue, those dominated by L. iners (CST II) in yellow, and those women with highly diverse vaginal microbiota (CST IV) are circled in green. Axes represent eigenvalues, a metric for which magnitude indicates the amount of variation captured in the PCoA axis. (C) A cluster dendrogram was calculated from Bray–Curtis dissimilarities and used to visualize clustering of the vaginal microbiota. Three clusters were observed using the gap statistic (Fig. S5A) and, in general, the previously described CSTs clustered together within the dendrogram. CST I (blue), L. cripatus dominant; CST II (yellow), L. iners dominant; CST IV (green), highly diverse. Asterisk indicates resolved to bacterial genus.

Sex workers on DMPA have low estrogen, vaginal glycogen and α-amylase. (A) To ensure sex workers on DMPA had detectable amounts of MPA in the circulation, plasma MPA was quantified in all study participants using ELISA. MPA was significantly higher in the sex workers in the DMPA group compared with the OCP and no hormone groups (Kruskal–Wallis test; P≤0.0001). (B) Sex workers on DMPA had significantly lower circulating estradiol than those not taking hormonal contraceptives (one-way ANOVA; P≤0.05). (C) Sex workers on DMPA also had significantly lower circulating progesterone than those on OCPs and not on hormonal contraceptives (Kruskal–Wallis test; P≤0.05). (D-G) As the diverse vaginal microflora can strongly influence vaginal carbohydrates in CVLs and overshadow the effect of hormones on these types of parameters (Moncla et al., 2016, 2015), we restricted our analysis of vaginal glycogen and α-amylase to sex workers with Nugent scores ≤3. (D) Vaginal glycogen was significantly lower in the vaginal lavage of sex workers on DMPA (N=16) compared with those not on hormonal contraceptives (proliferative phase of the menstrual cycle, N=18; unpaired t-test; P=0.043). (E) Similarly, α-amylase was significantly less abundant (Mann–Whitney; P=0.0095) in the vaginal lavage of sex workers on DMPA (N=16) compared with sex workers who were not on hormonal contraceptives (proliferative phase of the menstrual cycle, N=16). (F) Sex workers on OCPs had the highest levels of free vaginal glycogen, and significantly more free vaginal glycogen than the sex workers on DMPA (N=13, 16, respectively; P=0.008). (G) The quantity of α-amylase in the CVL supernatants of sex workers on OCPs versus those on DMPA verged on significance (N=13, 16, respectively; P=0.11). *P≤0.05, **P≤0.01, ****P≤0.0001. Data are mean±s.e.m.

DMPA is associated with lower vaginal glycogen and increased HIV-1 susceptibility in humanized mice. (A) To determine whether a subcutaneous injection of DMPA in the nape of the mouse neck could induce circulating concentrations similar to those observed in women, we administered 1 (N=7) or 2 mg (N=8) of DMPA or saline (control, N=5) to NRG mice (background strain of the humanized mice), collected peripheral blood by cardiac puncture 1 or 3 weeks later, and quantified MPA in the serum using ELISA. (B) Glycogen was quantified in the vaginal homogenate of humanized mice (controls, no hormonal treatment, N=10) and those treated with DMPA (N=8). Vaginal glycogen was significantly lower in the humanized mice treated with DMPA (Mann–Whitney U-test; P=0.017). (C) Control humanized mice (no hormonal treatment, N=20) and humanized mice given 2 mg DMPA (N=22) were challenged intravaginally with HIV-1. The infection rate in DMPA-treated humanized mice (77%) was higher than that in untreated control humanized mice (35%) (χ2; P=0.014). (D) The proportion of DMPA-treated humanized mice that became infected with HIV-1 was significantly greater (χ2; P=0.014) than the proportion of untreated control humanized mice that became infected following intravaginal challenge. N/A, not applicable. *P≤0.05, **P≤0.01, ***P≤0.001. Data are mean±s.e.m.

Proposed mechanism linking DMPA to changes in vaginal microenvironment and HIV-1 susceptibility in Kenyan sex workers. Taken together, our results suggest that estrogen (either from endogenous sources or oral contraceptive pills) is associated with greater quantities of vaginal glycogen and α-amylase, and minimal bacterial diversity within the vaginal microbiota of sex workers. This stable, uniform microbiota is not associated with susceptibility to HIV-1, albeit via incompletely understood mechanisms. In contrast, the hypo-estrogenism resulting from use of MPA lowers key metabolic products (i.e. glycogen, and α-amylase) that can be used by certain protective bacterial species, and the change in substrates allows other bacteria to colonize the vaginal microbiota, in effect increasing bacterial diversity. Diversity of the vaginal microbiota may contribute to enhanced susceptibility to HIV-1 in sex workers by an unknown mechanism that is independent of inflammatory cytokines and/or enhanced T cells.

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